The mechanisms of acute ethanol depression of myocardial contractility were examined in intact hearts, isolated cardiac muscle and in suspensions in individual isolated cardiac myocytes. In the isolated, rat heart, ethanol (0.75 - 6.0 vol%) caused a concentration-dependent decline in developed pressure without a change in ATP (adenosine triphosphate), phosphocreatine, inorganic phosphate, or pH measured by NMR techniques. The functional decline could be rapidly and completely reversed by perfusing the ethanol-free solution and significantly, although not completely, reversed by increasing perfusate calcium to 4 mM. In single rat cardiac myocytes and myocyte suspensions the acute effects of ethanol (1-5%) on cytosolic [Ca2+] (Cai) transient and contraction and on sarcoplasmic reticulum (SR) calcium content were examined. During stimulation at 1 Hz ethanol decreased the amplitude of the Cai transient and decreased the amplitude of contraction. The Cai transient amplitude was decreased to a lesser extent than the contraction amplitude. In myocyte suspensions, ethanol caused a concentration dependent initial increase in Cai and a subsequent depletion of SR calcium content, manifest as a diminution in the Cai increase elicited by caffeine in the presence of extracellular EGTA and zero added calcium. Thus, in rat cardiac myocytes high ethanol concentrations induces SR calcium release and depletes the SR of calcium, attenuates the Cai transient elicited by electrical stimulation and alters the myofilament calcium interaction possibly due to a decrease in myofilament calcium sensitivity. These combined effects contribute to the high concentration ethanol depression of contraction. In additional studies in isolated ferret cardiac muscle the threshold ethanol concentration (0.15=0.05%) which produced a 10% decline in contractile force did not change the transient. This decline in contraction strength with no change in Cai transient suggests that the negative inotropic effect of low concentrations of ethanol is due to an altered myofilament response to calcium.